Isolated ductal fluid sample

ABSTRACT

A sample for diagnosis of breast cancer can be prepared by isolating a ductal fluid sample from one duct of a breast of a patient. The isolated ductal fluid is not mixed with ductal fluid from any other duct of the breast. Generally the target duct is not spontaneously discharging. The isolated ductal fluid sample can be examined to determine the presence or absence of a marker associated with cancer or pre-cancer. An isolated ductal fluid sample not mixed with ductal fluid from any other duct of the breast permits identification of the duct which is diseased and provides increased sensitivity for existing diagnostic and analytic techniques.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application no.09/625,399 filed Jul. 26, 2000, which is a continuation-in-part ofapplication no. 09/502,404, filed on Feb. 10, 2000, which was acontinuation-in-part of application no. 09/313,463, filed on May 17,1999. This application is also a continuation-in-part of application no.09/473,510, filed on Dec. 28, 1999. This application also claims thebenefit under 37 CFR 1.78 of provisional application 60/166,100 filed onNov. 17, 1999. The full disclosures of each of the prior applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] For several decades significant members of the medical communitydedicated to studying breast cancer have believed and shown that thecytological analysis of cells retrieved from nipple discharge from thebreast milk ducts can provide valuable information for identifyingpatients at risk for breast cancer. Papanicolaou himself contributed tothe genesis of such a possibility of a “Pap” smear for breast cancer byanalyzing the cells contained in nipple discharge. See Papanicolaou etal, “Exfoliative Cytology of the Human Mammary Gland and Its Value inthe Diagnosis of Cancer and Other Diseases of the Breast” Cancer (1958)March/April 377-409. See also Petrakis, “Physiological, biochemical, andcytological aspects of nipple aspirate fluid”, Breast Cancer Researchand Treatment 1986; 8:7-19; Petrakis, “Studies on the epidemiology andnatural history of benign breast disease and breast cancer using nippleaspirate fluid” Cancer Epidemiology, Biomarkers and Prevention (Jan/Feb1993) 2:3-10; Petrakis, “Nipple Aspirate Fluid in epidemiologicalstudies of breast disease”, Epidemiologic Reviews (1993) 15:188-195.More recently, markers have also been detected in nipple fluid. SeeSauter et al, “Nipple aspirate fluid: a promising non-invasive method toidentify cellular markers of breast cancer risk”, British Journal ofCancer 76(4): 494-501 (1997). The detection of CEA in fluids obtained bya nipple blot is described in Imayama et al. (1996) Cancer 78:1229-1234. Further, an intraductal aspiration method for cytodiagnosisin situations of spontaneous nipple discharge (Hou et al, ActaCytologica 2000 v. 44:1029-1034) describes use of intraductal aspirationto collect specimens from spontaneously discharging ducts in order tomake a cytodiagnosis.

[0003] Breast cancer is believed to originate in the lining of a singlebreast milk duct; and additionally the human breast is believed tocontain from 6 to 9 of these ducts. See Sartorius, JAMA 224 (6): 823-827(1973). Sartorius describes use of hair-like single lumen catheters thatare inserted into breast ducts using an operating microscope and theducts were flushed with saline solution as described in Cassels, D Mar.20^(th), 1973, The Medical Post, article entitled “New tests may speedbreast cancer detection”. After the fluid was infused, the catheter wasremoved because it was too small to collect the fluid, the breast wassqueezed and fluid that oozed onto the nipple surface was removed fromthe surface by a capillary tube. Similarly, Love and Barsky,“Breast-duct endoscopy to study stages of cancerous breast disease”,Lancet 348(9033): 997-999, 1996 describes cannulating breast ducts witha single lumen catheter and infusing a small amount of saline, removingthe catheter and squeezing to collect the fluid that returns on thenipple surface. The use of a rigid 1.2 mm ductoscope to identifyintraductal papillomas in women with nipple discharge is described inMakita et al (1991) Breast Cancer Res Treat 18: 179-188. It would beadvantageous to collect the ductal fluid from within the duct and sofacilitate duct-specific analysis.

SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide a method forpreparing a sample for use in diagnosis of breast cancer or pre-cancer.

[0005] It is another object of the invention to provide an isolatedductal fluid sample suitable for analyzing breast cancer and pre-cancer.

[0006] It is yet another object of the invention to provide a method foranalyzing breast markers or epithelial cells.

[0007] These and other objects of the invention are provided by one ormore of the emobidments described below. In one embodiment a method isprovided for preparing a sample for use in the diagnosis of breastcancer or pre-cancer. A ductal fluid sample is isolated from one duct ofa breast of a patient. The isolated ductal fluid is not mixed withductal fluid from any other duct of the breast.

[0008] According to another emobidment of the invention an isolatedductal fluid sample is provided. The sample is collected from a breastduct in a breast. The isolated ductal fluid is not mixed with ductalfluid from any other breast duct.

[0009] According to still another embodiment of the invention a methodis provided for analyzing breast markers or epithelial cells. Thepresence or absence of a marker in an isolated ductal fluid sample isdetermined. The sample is collected from a breast duct in a breast. Theisolated ductal fluid not mixed with ductal fluid from any other breastduct.

[0010] The present invention thus provides the art with improved samplesand sampling techniques for diagnosing and prognosing breast cancer andpre-cancer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0011] The following preferred embodiments and examples are offered byway of illustration and not by way of limitation.

[0012] The invention comprises an isolated ductal fluid sample collectedfrom a breast duct in a breast, the fluid not mixed with ductal fluidfrom any other breast duct. The isolated ductal fluid sample can be asample from a non-discharging breast duct. A non-discharging duct is abreast duct that is not spontaneously discharging fluid or material,i.e., a duct which is not leaking fluid to the nipple surface.Spontaneously discharging ducts discharge fluid of various coloration.The spontaneous discharge itself is a warning sign usually requiringfurther investigation, such as, mammography, ductoscopy, and/orgalactography. The present invention provides an isolated ductal fluidsample from a non-discharging duct, i.e., a ductal fluid and/or materialsample, a portion of which would not otherwise have contacted the nipplesurface. However, the isolated ductal fluid sample may also be from adischarging duct, provided the sample collected is not mixed with ductalfluid from any other duct.

[0013] The isolated ductal fluid sample can be examined for the presenceof a marker, the absence of a marker, or the state or quality of aparticular marker. The markers can comprise those detailed herein andrelated markers that indicate the status or condition of the breast. Themarker status can be used to identify pre-cancer or cancer of thebreast. The ductal fluid sample is collected from one duct of a breastof a patient. Ductal fluids may be collected from multiple ducts of abreast or from ducts in both breasts of a patient, e.g., in sequence,provided the fluid and material from each duct is kept separate foranalysis from the other ducts. The ducts are also marked or otherwiseidentified so that follow-up and/or treatment of a duct that indicatesthe need for treatment can be conducted. The ductal fluid sample whencollected or provided is not mixed with ductal fluid from any other ductof the breast.

[0014] The number of epithelial cells in a ductal fluid sample mayrange, for example, from a few to a hundred, to several hundred, toseveral thousand, and up to tens of thousands, e.g., 20,000 to 100,000or more cells. At least ten epithelial cells are required to designatean isolated sample as adequate for analysis of the cells. An isolatedductal fluid sample can have 10 or more cells for analysis, and possiblya single clump of cells or more than one clump. A clump comprises aplurality of cells, generally at least about 4 to about 6 cells are in aclump, and the clump can comprise more cells than 6. Samples with one ormore clumps can also include individual cells that are distinct from theclump(s). Thus, an isolated sample retrieved by infusing fluid into theduct and collecting the infused fluid mixed with the ductal fluid canprovide multiple cells and one or more cell clumps for analysis. Theadvantage of cell clumps is that the clumping provides a framework foranalysis of cell-cell interaction or a cell-to-cell relationship that inturn provides information about the status of the cells themselves. Theinvention provides the a ductal fluid sample comprising sufficientductal epithelial cells from a breast duct for an analysis of the breastin which the duct is located. Insufficient ductal epithelial cells in asample means that a cytological analysis of those cells can not beperformed, or that the accuracy of the cytological analysis iscompromised. The method of the invention and the composition providesamples from single breast ducts that can be analyzed because thesamples so isolated contain sufficient material for an adequate analysisto be made. Ductal samples can comprise markers present in the fluid inaddition to cells, i.e., molecules present in the cells collected and/orin the extracellular material retrieved from the breast duct. Anadvantage provided by the invention is that many more cells than havebeen previously collected are collectable and an accurate cytologicalanalysis can therefore be made of the sample.

[0015] Relatively undisrupted cells and clumps can be analyzed toprovide information on the cellular status in the breast duct from whichthe sample was collected. Further, collection of the ductal fluid fromthe breast duct provides enough cells and/or other material from theduct to provide a useful analysis of the condition of the breast. Thisis largely due to the fact that collection of the ductal fluid, cellsand other material by infusing saline or other biocompatible wash fluidand collecting the wash fluid mixed with the ductal material results incollection of sufficient fluid and material for analysis. Suction may beapplied to the lumen in the duct to facilitate collection of the ductalfluid and material once the duct has been filled with wash fluid (inorder to prevent collapse of the ductal walls); reinfusion of wash fluidcan follow in order to prevent collapse of the ductal walls and providethe opportunity for a second or subsequent intraductal aspiration and/orretrieval. Squeezing and massaging the breast may also be used inconcert with infusion and collection procedures. The amount of materialthat is sufficient for analysis in a sample is at least one ductalepithelial clump up to at least 10 ductal epithelial clumps or more—eachclump having from at least 4 to 6 ductal epithelial cells. For example,the sample from a non-discharging or discharging breast duct may have atleast from 10 to 20 ductal epithelial cells, 20 to 50, 50 to 100, 100 to1000, 1000 to 10,000, 10,000 to 50,000, or 50,000 to 100,000 ductalepithelial cells. The ductal epithelial cells may be present eitherindividually or in clumps or both. Insufficient samples might includesamples with less than 10 epithelial cells. An evaluation ofinsufficiency may be contingent on whether the cells are clumped or not.

[0016] The method of the invention is preparing a sample for use indiagnosis of breast cancer or pre-cancer comprising isolating a ductalfluid sample from one duct of a breast of a patient. The isolated ductalfluid is not mixed with ductal fluid from any other duct of the breast.The method can further include examining the isolated ductal fluidsample to determine the presence or absence of a marker. The duct fromwhich the ductal fluid is isolated can be a duct that is notspontaneously discharging fluid. The marker for analysis can be selectedfrom any known and useful markers for a breast condition, includingpre-cancer and/or cancer markers, and further optionally includingmarkers listed herein.

[0017] The isolated fluid sample can be examined to determine thepresence of a marker. The presence of any marker, the absence of anymarker, or the quality or state of any marker can be analyzed orexamined. Particularly, the markers listed herein, and related forms orspecies of the markers listed herein are contemplated. The presence,absence or state of more than one marker can be examined. Markers can beexamined in conjunction with ductal epithelial cell cytologicalanalysis. The markers can be, for example, intracellular, nuclear,cytoplasmic, cell-surface, secreted, or extracellular markers. Themarkers can include any markers described in co-owned, co-pending,parent applications 09/625,399 filed Jul. 26, 2000, applicationno.09/502,404, filed on Feb. 10, 2000, and application no. 09/313,463,filed on May 17, 1999, hereby incorporated by reference in theirentirety.

[0018] Examination of the ductal fluid for a marker can comprisedetermining absorption of a marker molecule by abnormal cells in thefluid. For example, absorption of iodide or a like molecule by cells ina ductal fluid sample can be measured. Examination of the ductal fluidfor a marker can comprise analysis or examination of a quality and/orstate of nucleic acid for such characteristic changes from the normalstate as, for example, a loss of heterozygozity. Examination of theductal fluid can comprise examining the fluid for the absence of amarker; especially where the marker is present in normal ductal fluid ina predetermined quantity in the population, and standards are set forbenchmarks indicating a particular condition in the breast (ie.,pre-cancer or cancer, or their various sub-categories). Examination ofthe ductal fluid can comprise examining the ductal fluid for thepresence or absence of two or more markers, including examining two ormore markers for their state or quality, and including absorption of amarker, or loss of heterozygosity in the DNA of the cells retrieved fromthe isolated sample.

[0019] For example, the markers in this latter case can comprise DNAcontent, p53 gene or gene product, and G-actin or a nucleic acidencoding a polypeptide comprising at least a portion of G-actin. Thus,for example, examining the ductal fluid can comprise examining the fluidfor the presence of at least one marker and the absence of at least onemarker, e.g., examining the ductal fluid for the presence of an oncogeneor its gene product, and examining the ductal fluid for the absence of atumor suppressor molecule normally present in a given range or quantityin normal breast duct fluid or breast tissue. As an example, the ductalfluid can be examined for markers comprising such parameters as DNAcontent of the ductal epithelial cells, the absence or lowered levels ofp53 gene or its gene product, and the presence of G-actin protein,polypeptide, or portion thereof, or a nucleic acid encoding a G-actinprotein or polypeptide or portion thereof, e.g., as described in Rao etal, Cancer Epid, Biomarkers & Prevention, 1993 v. 7:1027-1033.

[0020] Preparing an isolated ductal fluid sample can comprise accessingthe duct with a ductal access tool and collecting the ductal fluidsample while the tool remains in the duct. Having the tool remain in theduct for fluid infusion and fluid collection also ensures that theductal fluid and ductal material collected are collected from a singleduct not mixed with fluid or material from any other duct. Wash fluidinfusion is used in the cases where the duct is not spontaneouslydischarging fluid so that the duct is filled or partially filled, thewash fluid mixes with ductal fluid and ductal contents, and retrieval ofthe mixed fluids comprises retrieval of a sample having sufficient cellsand/or other markers for analysis of the condition of the duct fromwhich the sample is taken. Previously the usefulness of ductal fluidretrieved by other means has been hampered by insufficient material orcells for analysis in the retrieved samples and/or not being able toidentify the specific duct to which abnormal cells or other findings canbe attributed. Since most breast cancers begin in a single, isolated,milk duct of a breast, the identification of a specific duct as abnormal(i.e., cancerous or pre-cancerous) is extremely useful, especially inconcert with sufficient information from the isolated fluid sample inorder to make a diagnosis. Collection of the isolated fluid sample canbe facilitated by fluid infusion into the duct and collection of thewash fluid that has been infused mixed with ductal fluid and otherductal contents including ductal epithelial cells and other markers. Thecollection through the accessing lumen is facilitated after wash fluidinfusion by a number of techniques that can be used together orseparately and which are not limited to squeezing the breast, massagingthe breast, applying negative pressure on the lumen to pull-up fluidinto the lumen and/or collection receptacle, and using an additive inthe wash fluid that delays or inhibits absorption of the fluid into theductal walls (thereby keeping more fluid in the duct to be retrieved).Many of these techniques and tools for practicing these techniques aredescribed in co-owned U.S. Ser. No. 09/067,661, U.S. Ser. No.09/301,058, PCT US99/09141, U.S. Ser. No. 09/313,463, U.S. Ser. No.09/473,510, PCT US99/31086 herein incorporated by reference in theirentirety.

[0021] By the procedure of ductal lavage, ductal epithelial cells thatline the walls of the ductal lumen are washed out of the duct. Lavage orwash fluid is infused into the duct, and the lavage fluid mixed withductal fluid is collected. Lavage is described in copending and co-ownedapplications including 09/067,661, 09/301,058, PCT US99/09141,60/122,076, 09/313,463, 60/143,359, and U.S. Ser. No. 09/473,510, allincorporated by reference in their entirety. Suction can be applied tothe tool accessing the ductal lumen in order to retrieve a maximumamount of cells and/or fluid. Lavage or wash fluid can be infused intothe duct, and collected. Suction can be applied to the tool accessingthe ductal lumen in order to retrieve a maximum amount of cells and/orfluid. The duct can be flushed by infusing saline into the duct untilresistance is met, applying pressure and/or squeezing the breast, e.g.,particularly at the base of the breast, and capturing the fluid thatmoves up through the duct after the pressure is applied. Flushing cancontinue by infusing more saline and applying more pressure.

[0022] In order to retrieve cells and ductal material sufficient foranalysis of a single non-discharging breast duct and a correspondingdiagnosis, a non-discharging duct can be accessed by a tool capable ofinfusing wash fluid and also capable of collecting the ductal fluidmixed with wash fluid while the tool remains in the duct as describedherein. Thus, ductal fluid can be retrieved by a medical tool, e.g., acatheter or a cannula, placed into the duct to infuse wash fluid toretrieve a mixture of wash fluid and ductal fluid from the duct withoutremoval of the tool. Thus, by the method of the invention, the toolremains indwelling while wash fluid is infused and wash fluid mixed withductal fluid (comprising cells and cellular material, etc.) iscollected. The fluid from the breast duct can contain ductal epithelialcells, including cells of a stage considered to be pre-cancerous orcancerous as described, and may also contain various molecules eitherconnected to the cells or separate from them that may be used asmarkers. Either presence or absence or decrease or increase relative toa normal or benign control amount can indicate cancer or pre-cancer.

[0023] The method is practiced by providing a ductal fluid sample fromat least one duct of a breast of the patient. Providing the ductal fluidsample can be accomplished by obtaining the sample from the breast or byreceiving a sample that had been previously obtained. For example, alaboratory can receive a ductal fluid sample from a patient or apractitioner, and the laboratory can be directed to make an analysis ofthe sample. The isolated fluid is collected by some available techniqueincluding, for example, ductal lavage of a single duct. In general,collection of isolated ductal fluid not mixed with ductal fluid fromanother duct of the breast can be accomplished by accessing the ductwith a breast duct access tool that infuses fluid and collects ductalfluid mixed with the infused fluid, while the tool remains in the duct.Also, the collection tube can be marked and the duct can be marked sothat the analysis of the fluid is traceable to one duct which can bere-identified and re-accessed if appropriate.

[0024] The marker may be a nucleic acid or protein form of the marker.For example, the marker may be “X”, and either a nucleic acid sequenceencoding at least a portion of X, or a gene product at least a portionof protein or polypeptide X can be determined or measured. The markermay also be a non-nucleic acid or a non-amino acid molecule, such as,for example, a small organic molecule, a lipid, a fat, a biologicallyformed organic acid or base, a carbohydrate or other sugar typemolecule, a polymer type molecule or a portion of such molecule, amoiety that characterizes a marker, for example a side chain on amarker, etc.

[0025] Thus, for example, the method of providing an isolated ductalfluid sample, and examining the sample for one or more markers cancomprise examining the sample for the presence, absence, or relativelevel of any one or more of the following markers:

[0026] 1. lysophosphatidic acid (LPA) or a lysophospholipid, or areceptor of lysophosphatidic acid, e.g., as described in Goetzl et al,Cancer Res 1999 Sep 15 v.59: 4732-7, Xu et al, Biochem J 1995 v.309:933-40, and Contos et al, Mol Pharmacol 2000 v.58: 1188-1196;

[0027] 2. palladin, a portion of palladin, or a nucleic acid encoding apolypeptide comprising at least a portion of paladin, e.g., as describedin Reuters Health News Aug. 7, 2000, and Parast and Otey, J Cell Biol2000 Aug. 7 v. 150:643-56;

[0028] 3. Lg, a portion of Lg, or a nucleic acid encoding a polypeptidecomprising at least a portion of Lg, e.g., as described in Ranganathanet al, J Steroid Biochem Mol Biol 1999 v.70: 151-8;

[0029] 4. E2F1, a portion of E2F1, or a nucleic acid encoding apolypeptide comprising at least a portion of E2F1, e.g., as described inKlein-Szanto et al Cancer Epid Biomarkers & Prevention 2000 v. 9:395-401;

[0030] 5. TIA 12/mac 25, a portion of TIA 12/mac 25, or a nucleic acidencoding a polypeptide comprising at least a portion of T1A12/mac 25,e.g., as described in Burger et al Oncogene 1998 v. 16:2459-67;

[0031] 6. MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid encoding apolypeptide comprising at least a portion of MAGUK/ZO-1, e.g., asdescribed in Hoover et al, Am J Pathol 1998 v.153: 1767-73;

[0032] 7. Repressor of estrogen receptor activity (REA), a portion ofREA, a nucleic acid encoding a polypeptide comprising at least a portionof REA, e.g., as described in Simon et al, Cancer Res 2000 v. 60:2796-9;

[0033] 8. prothymosin alpha (PTA), a portion of PTA, a nucleic acidencoding a polypeptide comprising at least a portion of PTA, e.g., asdescribed in Domineguez et al, Br J of Cancer 2000 v. 82:584-590; andMagdalena et al, Br J Cancer 2000 v. 82:584-590;

[0034] 9. TNF-related apoptosis-inducing ligand (TRAIL), a nucleic acidencoding a polypeptide comprising at least a portion of TRAIL, e.g., asdescribed in Griffith et al, J lmmunol 2000 v. 165:2886-94; andHerrnring et al Histochem Cell Biol 2000 v. 113:189-94;

[0035] 10. BU101 protein, a nucleic acid encoding a polypeptidecomprising at least a portion of BU0101, e.g., as described in WO98/07857;

[0036] 11. c-raf kinase, a portion of c-raf kinase, a nucleic acidencoding a polypeptide comprising at least a portion of c-raf kinase,e.g., as described in E1-Ashry et al, Oncogene 1997 v.15: 423-35, andCallans et al, Ann Surg. Oncol 1995 v.2: 38-42;

[0037] 12. CD66a, a portion of CD66a, a nucleic acid encoding apolypeptide comprising at least a portion of CD66a, e.g., as describedin Huang et al Anticancer Res. 1998 v.18: 3203-12;

[0038] 13. KL-1, a portion of KL-1, a nucleic acid encoding apolypeptide comprising at least a portion of KL-1, e.g., as described inOkumura et al, Jpn J Clin Oncol 1998 v.28: 480-5;

[0039] 14. cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, anucleic acid encoding a polypeptide comprising at least a portion of CAM5.2, e.g., as described in Okumura et al, Jpn J Clin Oncol 1998 v.28:480-5;

[0040] 15. leptin, a portion of leptin, a nucleic acid encoding apolypeptide comprising at least a portion of leptin, e.g., as describedin Tessitore et al, Int J Mol Med 2000 v.5: 421-6;

[0041] 16. Bcl-2 gene product, at least a portion of Bcl-2 gene productor polypeptide, a nucleic acid encoding a polypeptide encoding at leasta portion of Bcl-2 gene product, e.g., as described in Krajewski et alEndocr Relat Cancer 1999 v.6: 29-40, and Castiglione et al AnticancerRes. 1999 v.19:4555-63, Simony-Lafontaine et al, 2000 v.82: 1958-66;

[0042] 17. nuclear matrix 23(nm23), a portion of nm23, a nucleic acidencoding a polypeptide comprising at least a portion of nm23, e.g., asdescribed in Vazquez-Ramirez et al, Pathol Res Pract 2000 v.196: 553-9,Cipollini et al, Cancer Genet Cytogenet 2000 v. 121:181-5;

[0043] 18. an apotosis-related protein, a portion of said protein, anucleic acid encoding a polypeptide comprising at least a portion of theapoptosis-related protein, e.g., as described in Dowsett et al, EndocrRelat Cancer 1999 v.6: 25-8:

[0044] 19. lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acidencoding a polypeptide comprising at least a portion of lipocalin NGAL,e.g., as described in Stoesz et al, Int J Cancer 1998 v.79: 565-72;

[0045] 20. thymosin beta-15, a portion of thymosin beta-15, a nucleicacid encoding a polypeptide comprising at least a portion of thymosinbet-15, e.g., as described in U.S. Pat. No. 5,663,071 and WO 97/48805;

[0046] 21. tumor amplified kinase STK15 (also BTAK and aurora2), atleast a portion of STK15, a nucleic acid encoding at least a portion ofSTK15; e.g., as described in Zhou et al, Nat Genet 1998 v.20:189-93;

[0047] 22. complement regulatory protein CD46, a portion of CD46, anucleic acid encoding at least a portion of CD46; as described e.g., inThorsteinsson et al APMIS 1998 v.106:869-78;

[0048] 23. complement regulatory protein CD59, a portion of CD59, anucleic acid encoding at least a portion of CD59; as described e.g., inThorsteinsson et al APMIS 1998 v.106:869-78;

[0049] 24. a nucleic acid encoding a portion of an FHIT gene, e.g., asdescribed in Huiping et al, Eur J Cancer 2000 v.36: 1552-7, Gatalica etal, Cancer 2000 v.88: 1378-83, Ahmadian et al, Cancer Res 1997 v.57:3664-8, and Campiglio et al, Cancer Res 1999 v. 59:3866-9;

[0050] 25. loss of heterozygosity (LOH), e.g., as described in Lingingeret al, Mod Patbol 1998 v. 11:1151-9; and Larson et al, Am J Pathol 1998v. 152:1591-8;

[0051] 26. LOH at an FRA3B site, e.g., as described in Ahmadian et al,Cancer Res 1997 v. 57:3664-8;

[0052] 27. MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding atleast a portion of MRP-1/CD9, e.g., as described in van denHeuvel-Eibrink et al, Int J Clin Pharmacol Ther 2000 v.38:94-110, Huanget al Am J Pathol 1998 v 0.153: 973-83, Miyake et al Cancer Res 1996v.56:1244-9, and Miyake et al Cancer Res. 1995 v.55: 4127-31;

[0053] 28. KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding atleast a portion of KAI1/CD82, e.g., as described in Huang et al Am JPathol 1998 v.153: 973-83;

[0054] 29. TMS-1, a portion of TMS-1, a nucleic acid encoding apolypeptide comprising at least a portion of TMS-1, for example asdescribed in McConnell and Vertino, Cancer Res. 2000 Nov.15;60(22):6243-7; Conway et al, Cancer Res 2000 Nov. 15;60(22):6236-42;and Grossman et al, J Exp Biol 2000 v.203: 447-57;

[0055] 30. at least a portion of breast cancer associated gene (BRCA),e.g., as described in Seances et al, Soc. Biol Fil 1998 v. 192:35-40,and Deng and Brodie, Bioessays 2000 v.22: 728-37;

[0056] 31. absorption of a marker (e.g., iodide), e.g., as described inDe La Vieja et al, Physiol Rev 2000 v.80: 1083-105, Tazebay et al, NatMed 2000 v. 6:871-8;

[0057] 32. Fibroblast growth factor (FGF) protein, a portion of an FGFprotein or polypeptide, a nucleic acid encoding at least a portion of anFGF protein or polypeptide, e.g., as described in Femig et al, CancerTreat Res 1991 v.53: 47-78, and De Benedetti and Harris, Int J BiochemCell Biol 1999 v.31: 59-72;

[0058] 33. Vascular endothelial growth factor (VEGF) protein, a portionof a VEGF protein or polypeptide, a nucleic acid encoding at least aportion of a VEGF protein or polypeptide e.g., as described inGasparini, Oncologist 2000; 5 suppl 1:37-44;

[0059] 34. Insulin-like growth factor -1 (IGF-1 protein, a portion of anIGF-1 protein or polypeptide, a nucleic acid encoding at least a portionof an IGF-1 protein or polypeptide e.g., as described in Pollack, Eur JCancer 2000 v. 36:1224-8;

[0060] 35. Maspin protein, a portion of a maspin protein or polypeptide,a nucleic acid encoding at least a portion of a maspin protein orpolypeptide, e.g., as described in Sager et al, Adv Exp Med Biol 1997v.425: 77-88.

[0061] 36. CDw60 protein, a portion of CDw60 protein or polypeptide, anucleic acid encoding at least a portion of a CDw60 protein orpolypeptide, e.g., as described in Gocht et al, Histochem J 2000 Jul;32(7):447-56.

[0062] 37. Mammary expressed enzymes (e.g., cytochrome P450s,catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S-transferases, N-acetyltransferases, and sulfotransferases)a nucleic acid encoding at least a portion of a mammary expressedenzyme, e.g., as described in Williams and Phillips, Cancer Res 2000 Sep1:60(17):4667-77.

[0063] 38. Mammastatin protein or polypeptide (47 kD and/or 65 kD), anucleic acid encoding at least a portion of a mammastatin protein orpolypeptide, e.g., as described in Ervin et al, Science 1989; 244(4912);1585-7.

[0064] 39. Kallikrein 6 (zyme/protease M/neurosin) protein orpolypeptide (hK6), a nucleic acid encoding at least a portion of an hK6protein or polypeptide, e.g., as described in Diamandis et al, ClinBiochem 2000 Oct; 33(7):579-583; Diamandis et al, Clin Biochem 2000 Jul;33(5):36975; Yousef et al, Genomics 2000 Nov. 1;69(3):331-41.

[0065] As discussed, the cells collected can comprise ductal epithelialcells and the ductal fluid collected can comprise molecular and cellularmaterial. The collected cells and fluid and fluid components can beanalyzed, e.g., as described or suggested herein. Fluid collected fromthe milk ducts, can include constituents of biological fluids, e.g.,those typically found in breast duct fluid, e.g., water, cells, cellularmarkers, molecular markers, nucleic acids, proteins, cellular debris,salts, particles or organic molecules. These constituents can beanalyzed by any appropriate method depending on the marker and thediagnostic purpose. In addition, any of the cells of the duct can beanalyzed for morphological abnormalities in cell components, including,e.g., morphological abnormalities of the nucleus, cytoplasm, Golgiapparatus or other parts of a cell. Cell morphology can serve toestablish whether the ductal epithelial cells are normal (i.e., notpre-cancerous or cancerous or having another noncancerous abnormality),pre-cancerous (i.e., comprising hyperplasia, a typical ductalhyperplasia (ADH) or low grade ductal carcinoma in situ (LG-DCIS)) orcancerous (ie., comprising high grade ductal carcinoma in situ(HG-DCIS), or invasive carcinoma). Analysis of cell contents may serveto establish similar staging as established by morphology, capturinggenerally a progression of a pre-cancerous or cancerous condition in thecells.

[0066] Once the ductal fluid sample is retrieved from the breast it isexamined for the presence of a marker such as, for example a protein, apolypeptide, a peptide, a nucleic acid, a polynucleotide, an mRNA, asmall organic molecule, a lipid, a fat, a glycoprotein, a glycopeptide,a carbohydrate, an oligosaccharide, and a chromosomal abnormality, awhole cell having a marker molecule, a particle, a secreted molecule, anintracellular molecule, and a complex of a plurality of molecules asdescribed above. In addition, the marker may be capable ofdistinguishing between any two cytological categories consisting ofnormal, abnormal, hyperplasia, atypia, ductal carcinoma, ductalcarcinoma in situ (DCIS), ductal carcinoma in situ—low grade (DCIS-LG),ductal carcinoma in situ high grade (DCIS-HG), invasive carcinoma, atypical mild changes, a typical marked changes, a typical ductalhyperplasia (ADH), insufficient cellular material for diagnosis, andsufficient cellular material for diagnosis. These categories classifythe epithelial cells cytologically, and these classifications mayindicate either cancer or its precursors, or absence of cancer indicia.

[0067] Analysis of cell contents may serve to establish similar stagingas established by morphology, capturing generally a progression of apre-cancerous or cancerous condition in the cells. Thus the ductalepithelial cells may be analyzed for other markers, e.g., proteinmarkers, nucleic acid markers, particles, complexes, or biochemical ormolecular markers in the cells or on the cell surfaces or secreted bythe cell or for any marker providing evidence of neoplasia. The ductalepithelial cell can be derived from any part of the breast milk duct,including, e.g., the ductal lumen and/or the terminal ductal lobularunit (TDLU). Cells derived from the TDLU may also have similar stages asfound in other lumenal ductal epithelial cells not from the TDLUincluding, e.g., hyperplasia, atypia, in situ carcinoma, and invasivecarcinoma.

[0068] Once the wash fluid has been infused in the duct and the washfluid and ductal fluid is collected from a breast duct, the cellularmaterial can be separated and can be examined. The cellular material caninclude, e.g., substances selected from the group consisting of wholecells, cellular debris, proteins, nucleic acids, polypeptides,glycoproteins, lipids, fats, glycoproteins, small organic molecules,metabolites, and macromolecules. These materials may be found in thecell, on the cell surface or as material secreted from the cell andfound in fluid outside the cell. These materials may be synthesized by acell, or may be otherwise present in the fluid from the duct, e.g., asby-products or degradation products of molecules in the body. Cytology,or any other suitable method for analyzing the condition of the cellscan be used to examine whole cells. Other markers present in thecellular material, ductal fluid, or other material obtained from thebreast duct can be analyzed as is appropriate for the marker beingsought, including e.g., binding assays, immunohistochemistry, or usingother analytical techniques for distinguishing and identifyingbiological molecules obtained from biological material. Examining theductal fluid sample can also be performed to determine the presence of amarker comprising, for example, a protein, a polypeptide, a peptide, anucleic acid, a polynucleotide, an mRNA, a small organic molecule, alipid, a fat, a glycoprotein, a glycopeptide, a carbohydrate, anoligosaccharide, a chromosomal abnormality, a whole cell having a markermolecule, a particle, a secreted molecule, an intracellular molecule, ora complex of a plurality of molecules. Detection and analysis of theseclassifications of markers can be accomplished, for example, usingstandard assays for determining the presence of a particular marker ormarker classification and/or for example as described in Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. (Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 1989).

[0069] Examining the ductal fluid sample can comprise determining thepresence of a marker comprising RNA, DNA, protein, polypeptide, orpeptide form of a marker such as lysophosphatidic acid, alysophospholipid, paladin, a portion of palladin, a nucleic acidencoding a polypeptide comprising at least a portion of paladin, Lg, aportion of Lg, a nucleic acid encoding a polypeptide comprising at leasta portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding apolypeptide comprising at least a portion of E2F1, T1A12/mac 25, aportion of T1A12/mac 25, a nucleic acid encoding a polypeptidecomprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion ofMAGUK/ZO-1, a nucleic acid encoding a polypeptide comprising at least aportion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), aportion of REA, a nucleic acid encoding a polypeptide comprising atleast a portion of REA, prothymosin alpha (PTA), a portion of PTA, anucleic acid encoding a polypeptide comprising at least a portion ofPTA, c-raf kinase, a portion of c-raf kinase, a nucleic acid encoding apolypeptide comprising at least a portion of c-raf kinase, CD66a, aportion of CD66a, a nucleic acid encoding a polypeptide comprising atleast a portion of CD66a, KL-1, a portion of KL-1, a nucleic acidencoding a polypeptide comprising at least a portion of KL-1, celladhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acidencoding a polypeptide comprising at least a portion of CAM 5.2, leptin,a portion of leptin, a nucleic acid encoding a polypeptide comprising atleast a portion of leptin, Bcl-2 gene product, at least a portion ofBcl-2 gene product or polypeptide, a nucleic acid encoding a polypeptideencoding at least a portion of Bcl-2 gene product, nuclear matrix23(nm23), a portion of nm23, a nucleic acid encoding a polypeptidecomprising at least a portion of nm23, an apotosis-related protein, aportion of said protein, a nucleic acid encoding a polypeptidecomprising at least a portion of the apoptosis-related protein,comprises lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acidencoding a polypeptide comprising at least a portion of lipocalin NGAL,a nucleic acid encoding a portion of an FHIT gene, loss ofheterozygosity at an FRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, anucleic acid encoding at least a portion of MRP-1/CD9, KAI1/CD82, aportion of KAI1/CD82, a nucleic acid encoding at least a portion ofKAI1/CD82, at least a portion of breast cancer associated gene, TMS-1, aportion of TMS-1, a nucleic acid encoding a polypeptide comprising atleast a portion of TMS-1; at least a portion of breast cancer associatedgene (BRCA); absorption of a marker (e.g., iodide). fibroblast growthfactor (FGF) protein, a portion of an FGF protein or polypeptide, anucleic acid encoding at least a portion of an FGF protein orpolypeptide, vascular endothelial growth factor (VEGF) protein, aportion of a VEGF protein or polypeptide, a nucleic acid encoding atleast a portion of a VEGF protein or polypeptide, insulin-like growthfactor -1 (IGF-1 protein, a portion of an IGF-1 protein or polypeptide,a nucleic acid encoding at least a portion of an IGF-1 protein orpolypeptide; maspin protein, a portion of a maspin protein orpolypeptide, a nucleic acid encoding at least a portion of a maspinprotein or polypeptide, CDw60 protein, a portion of CDw60 protein orpolypeptide, a nucleic acid encoding at least a portion of a CDw60protein or polypeptide, mammary expressed enzymes (e.g., cytochromeP450s, catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S-transferases, N-acetyltransferases, and sulfotransferases)a nucleic acid encoding at least a portion of a mammary expressedenzyme, mammastatin protein or polypeptide (47 kD and/or 65 kD), anucleic acid encoding at least a portion of mammastatin protein orpolypeptide; kallikrein 6 (zyme/protease M/neurosin) protein orpolypeptide (hK6), and a nucleic encoding at least a portion of an hK6protein or polypeptide.

[0070] A level of the marker can be a presence relative to a normalcontrol or an absence relative to a normal control of a given marker.Increased or decreased amounts relative to such normal controls can alsobe determined. The normal control can be determined relative to theparticular patient, or relative to a patient population. In addition,the quality of the marker can be assessed. A quality of a marker can besuch changes as DNA mutation, or a quantity of mutations, adeterioration of chromosomal quality or quantity, degradation of aprotein, or a change in quantity of a nucleic acid or chromosome. Aquality can be an erosion of a molecule, particle, molecule or organellewith respect to a normal quality. A tumor suppressor, e.g., mammastatinmay be used as a marker where a reduction in the marker identifies acancerous or pre-cancerous condition in the breast.

[0071] Chromosomal abnormalities in ductal epithelial cells can alsoprovide information and act as a marker to identify cancer or pre-canceras described in Mark et al (1999) Cancer Genet Cytogenet 108:26-31;Lundlin and Mertens (1998) Breast Cancer Res Treat 51:1-15; Newsham(1998) Am J Pathol 153:5-9; Larson et al (1998) Am J Pathol 152:1591-8;Adelaide et al (1998) Genes Chromosomes Cancer 22:186-99; Fejzo et al(1998) Gene Chromosome Cancer 22:105-113; Dietrich et al (1998) HumPathol 12: 1379-82; Cavalli et al (1997) Hereditas 126:261-8; Adeyinkaet al (1997) Cancer Genet Cytogenet 97:119-21; Afify and Mark (1997)Cancer Genet Cytogenet 97:101-5; Brenner and Aldaz (1997) Prog Clin BiolRes 396: 63-82; Mark et al (1997) Ann Clin Lab Sci 27:47-56; and Fabianet al 1993 J. Cellular Biochemistry 17G: 153-16.

[0072] Standard assay procedures for identifying the markers can beused, including antibodies or other binding partners, labels, stains,pattern analysis (for cells and cell components), and in general anyother chemical or visual identification techniques.

[0073] The different categories of markers are tested differentlydepending on the category and possibly also on the location of themarker in the cell (for example, a cell surface protein might bedetected differently than a cytoplasmic or nuclear protein). Typically,assays comprising one or more of binding, coloration, precipitation,affinity column selection, in-situ binding, solution phase binding,nucleic acid probe labeling, protein probe labeling, polypeptide probelabeling, peptide probe labeling, and/or a combination or variation ofthese processes can be used. Standard procedures for conducting suchassays generally (e.g., ELISA, RNA or DNA probe hybridization, and otherbinding or other detection assays) are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. (Cold Spring HarborPress, Cold Spring Harbor, N.Y. 1989). Standard assay procedures foridentifying the markers can be used, including antibodies or otherbinding partners, labels, stains, pattern analysis (for cells and cellcomponents), and in general any other chemical or visual identificationtechniques.

[0074] In general, markers can be categorized nonexclusively, and oftenin overlapping categories, for example, protein expression, mRNAexpression, post-translational change in a protein and/or DNA change ina gene may all be used in concert or separately for the same or aplurality of markers to make a diagnosis.

[0075] Cytology, or any other suitable method for analyzing thecondition of the cells can be used to examine whole cells. Markerspresent in the cellular material, ductal fluid generally, or othermaterial obtained from the breast duct can be analyzed as is appropriatefor the marker being sought, including, e.g., binding assays,immunohistochemistry, or using other analytical techniques fordistinguishing and identifying biological molecules obtained frombiological material.

[0076] Once the ductal fluid is analyzed for one or more markers, thefluid may also be analyzed cytologically to determine the cytologicalstatus of the ductal epithelial cells and other cells. Cytologicalassays that can be performed on the cells retrieved from a duct or fromnipple aspirate can include e.g., assays described in King et al, JNat'l Cancer Inst (1983) 71:1115-21, Wrensch et al. (1992) Am. J Epidem.135: 130-141, Papanicolaou et al, (1958) Cancer, 11:377-409 and GoodsonWH & King EB, Chapter 4: Discharges and Secretions of the Nipple, THEBREAST: COMPREHENSIVE MANAGEMENT OF BENIGN AND MALIGNANT DISEASES (1998)2^(nd) Ed. vol 2, Bland & Kirby eds. W. B. Saunders Co, Philadelphia,Pa. pp. 51-74. For example, as described in Goodson and King (page 60) atypical hyperplasia presents as having cellular abnormalities, increasedcoarseness of the chromatin, and tendency for more single cells as wellas groups of cells. With regard to carcinoma in situ, Papanicolaou et aldescribed cellular abnormalities, e.g., nuclear abnormalities diagnosedby cytology of fluid from nipple secretions containing ductal cells. Thecytological examination of abnormal cells can also be conducted asdescribed in Sartorius etal (1977) J Natl Cancer Inst 59: 1073-1080. andKing et al, (1983)JNCI 71(6) 1115-1121. Atypia and carcinoma in situ arewidely characterized pathologically, as described in Page et al, (1998)Mod Pathol 11(2): 120-8. The ductal fluid can be analyzed by cytologicaltechniques by placing some of the fluid on a slide with a standardcytological stain and observing under a light microscope. The cells canbe studied for atypical growth patterns in individual cells and clustersof cells using published methods, including Mouriquand J, (1993) SKarger Pub, “Diagnosis of Non-Palpable Breast Lesions:Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic andPrognostic Implications of Cytology” (ISBN 3805557477); Kline TS and IK,Pub Igaku-Shoin Medical “Breast: Guides to Clinical Aspiration Biopsy”(LSBN 0896401596; Masood, American Society of Clinical Pathology: Nov.199S, “Cytopathology of the Breast” ISBN 0891893806; and Feldman PS,American Society of Clinical Pathology, Nov. 1984, “Fine NeedleAspiration Cytology and Its Clinical Applications: Breast and Lung” ISBN0891891846.

[0077] Other references that discuss cytological analysis and which giveguidance to an analysis of ductal epithelial cells derived from ductalfluid include Silverman et al, (Can FNA biopsy separate a typicalhyperplasia, carcinoma in situ, and invasive carcinoma of the breast?Cytomorphologic criteria and limitations in diagnosis, DiagnosticCytopathology) 9(6): 713-28, 1993; Masood et al, (Immunohistochemicaldifferentiation of a typical hyperplasia vs. carcinoma in situ of thebreast) Cancer Detection & Prevention. 16(4): 225-35, 1992; Masood etal, (Cytologic differentiation between proliferative andnonproliferative breast disease in mammographically guided fine-needleaspirates) Diagnostic Cytopathology.7 (6): 581-90, 1991; Masood S.,(Occult breast lesions and aspiration biopsy: a new challenge)Diagnostic Cytopathology. 9(6): 613-4, 1993; Masood S., (Prognosticfactors in breast cancer: use of cytologic preparations) DiagnosticCytopathology. 13(5): 388-95, 1995, Novak and Masood, (Nuclear groovesin fine-needle aspiration biopsies of breast lesions: do they have anysignificance?) Diagnostic Cytopathology. 18(5): 333-7, 1998; Sidawy etal, (Interobserver variability in the classification of proliferativebreast lesions by fine-needle aspiration: results of the PapanicolaouSociety of Cytopathology Study) Diagnostic Cytopathology. 18(2): 150-65,1998; Masood et al, (Automation in cytology: a survey conducted by theNew Technology Task Force, Papanicolaou Society of Cytopathology)Diagnostic Cytopathology. 18(1): 47-55, 1998; and Frykberg and MasoodCopeland EM 3d. Bland KI., (Ductal carcinoma in situ of the breast)Surgery, Gynecology & Obstetrics 177(4): 425-40, 1993.

[0078] The invention also provides systems for preparing a sample foruse in diagnosis of breast cancer or pre-cancer, the system comprising atool to retrieve ductal fluid from a breast duct and instructions foruse to isolate a ductal fluid sample from a duct, particularly anon-spontaneously discharging breast duct in order to determine thepresence of one or more markers. Materials and instructions may also beincluded in the system to determine the presence or absence of a markerin the isolated ductal fluid. Materials may be included to make acytodiagnosis of collected ductal epithelial cells. A cytologicalreading of the ductal epithelial cells collected with the infused washfluid is one type of marker which can be used for diagnosing a conditionin a breast duct. Instructions in the kit or system can include guidancefor interpreting cytological data and/or other marker data in order tomake a diagnosis. The systems or kits may include a ductal access tool,for example in order to retrieve the ductal fluid, e.g., especiallywhere it is preferred that the ductal fluid be identified as coming froma specific duct (so that the duct can be accessed later for treatmentand/or further monitoring). Methods for identifying thenon-spontaneously discharging duct may also be included in the system orkit. The instructions in the systems or kits can include directionsaccording to the methods of identifying breast cancer or pre-cancerdescribed herein, and possibly including any marker or markers or markerclassification group or groups that could be useful and/or are describedherein. The system or kit can include assay reagents for detecting themarker or markers. The system or kit may comprise a panel of reagentsfor detecting a plurality of markers either simultaneously orsequentially, or some other practical combination of testing modalities.The system or kit can also include indexes and parameters for making adiagnosis, depending on the marker or markers. The system or kit caninclude a container for the contents of the system or kit.

EXAMPLES

[0079] Retrieval of Ductal Fluid and Analysis of Markers in the Fluid

[0080] A patient is prepared for a ductal access procedure. Using aductal access tool, a duct on each breast is infused with sufficientwash fluid, and the wash fluid mixed with ductal fluid is collectedseparately from each accessed duct. The fluid in each duct that isaccessed is analyzed for the presence, absence or relative levels (ascompared to a predetermined normal level) of one or more of thefollowing markers using standard techniques: lysophosphatidic acid, alysophospholipid, paladin, a portion of palladin, a nucleic acidencoding a polypeptide comprising at least a portion of paladin, Lg, aportion of Lg, a nucleic acid encoding a polypeptide comprising at leasta portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding apolypeptide comprising at least a portion of E2F1, T1A12/mac 25, aportion of T1A12/mac 25, a nucleic acid encoding a polypeptidecomprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion ofMAGUK/ZO-1, a nucleic acid encoding a polypeptide comprising at least aportion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), aportion of REA, a nucleic acid encoding a polypeptide comprising atleast a portion of REA, prothymosin alpha (PTA), a portion of PTA. anucleic acid encoding a polypeptide comprising at least a portion ofPTA, TNF-related apoptosis-inducing ligand (TRAIL), a nucleic acidencoding a polypeptide comprising at least a portion of TRAIL, BU101protein, a nucleic acid encoding a polypeptide comprising at least aportion of BU101 , c-raf kinase, a portion of c-raf kinase, a nucleicacid encoding a polypeptide comprising at least a portion of c-rafkinase, CD66a, a portion of CD66a, a nucleic acid encoding a polypeptidecomprising at least a portion of CD66a, KL-1, a portion of KL-1, anucleic acid encoding a polypeptide comprising at least a portion ofKL-1, cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, anucleic acid encoding a polypeptide comprising at least a portion of CAM5.2, leptin, a portion of leptin, a nucleic acid encoding a polypeptidecomprising at least a portion of leptin, Bcl-2 gene product, at least aportion of Bcl-2 gene product or polypeptide, a nucleic acid encoding apolypeptide encoding at least a portion of Bcl-2 gene product, nuclearmatrix 23(nm23), a portion of nm23, a nucleic acid encoding apolypeptide comprising at least a portion of nm23, an apotosis-relatedprotein, a portion of said protein, a nucleic acid encoding apolypeptide comprising at least a portion of the apoptosis-relatedprotein, comprises lipocalin NGAL, a portion of lipocalin NGAL, anucleic acid encoding a polypeptide comprising at least a portion oflipocalin NGAL, thymosin beta-15, a portion of thymosin beta-15, anucleic acid encoding a polypeptide comprising at least a portion ofthymosin beta-15, a nucleic acid encoding a portion of an FHIT gene,loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion ofMRP-1/CD9, a nucleic acid encoding at least a portion of MRP-1/CD9,KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding at least aportion of KAI1/CD82, at least a portion of breast cancer associatedgene, TMS-1, a portion of TMS-1, a nucleic acid encoding a polypeptidecomprising at least a portion of TMS-1; at least a portion of breastcancer associated gene (BRCA); absorption of a marker (e.g., iodide).fibroblast growth factor (FGF) protein, a portion of an FGF protein orpolypeptide, a nucleic acid encoding at least a portion of an FGFprotein or polypeptide, vascular endothelial growth factor (VEGF)protein, a portion of a VEGF protein or polypeptide, a nucleic acidencoding at least a portion of a VEGF protein or polypeptide,insulin-like growth factor -1 (IGF-1 protein, a portion of an IGF-1protein or polypeptide, a nucleic acid encoding at least a portion of anIGF-1 protein or polypeptide; maspin protein, a portion of a maspinprotein or polypeptide, a nucleic acid encoding at least a portion of amaspin protein or polypeptide, CDw60 protein, a portion of CDw60 proteinor polypeptide, a nucleic acid encoding at least a portion of a CDw60protein or polypeptide, mammary expressed enzymes (e.g., cytochromeP450s, catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S-transferases, N-acetyltransferases, and sulfotransferases)a nucleic acid encoding at least a portion of a mammary expressedenzyme, mammastatin protein or polypeptide (e.g., 47 kD and/or 65 kD), anucleic acid encoding a mammastatin protein or polypeptide; kallikrein 6(zyme/protease M/neurosin) protein or polypeptide (hK6), and a nucleicacid encoding at least a portion of an hK6 protein or polypeptide.Cytodiagnosis of a sample of the collected ductal epithelial cells, asindividual cells and as cells in clumps is also made to support anyother marker data from the collected fluid and material.

[0081] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for preparing a sample for use in diagnosis of breast cancer or pre-cancer comprising: isolating a ductal fluid sample from one duct of a breast of a patient, said isolated ductal fluid not mixed with ductal fluid from any other duct of the breast.
 2. A method as in claim 1, further comprising: examining the isolated ductal fluid sample to determine the presence or absence of a marker.
 3. A method as in claim 1, wherein the duct from which the ductal fluid is isolated is not spontaneously discharging ductal fluid.
 4. A method as in claim 2, wherein the marker is selected from the group consisting of: lysophosphatidic acid, a lysophospholipid, paladin, a portion of palladin, a nucleic acid encoding a polypeptide comprising at least a portion of paladin, Lg, a portion of Lg, a nucleic acid encoding a polypeptide comprising at least a portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding a polypeptide comprising at least a portion of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, a nucleic acid encoding a polypeptide comprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion of MAGUKIZO-1, a nucleic acid encoding a polypeptide comprising at least a portion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), a portion of REA, a nucleic acid encoding a polypeptide comprising at least a portion of REA, prothymosin alpha (PTA), a portion of PTA, a nucleic acid encoding a polypeptide comprising at least a portion of PTA, c-raf kinase, a portion of c-raf kinase, a nucleic acid encoding a polypeptide comprising at least a portion of c-raf kinase, CD66a, a portion of CD66a, a nucleic acid encoding a polypeptide comprising at least a portion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding a polypeptide comprising at least a portion of KL-1, cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acid encoding a polypeptide comprising at least a portion of CAM 5.2, leptin, a portion of leptin, a nucleic acid encoding a polypeptide comprising at least a portion of leptin, Bcl-2 gene product, at least a portion of Bcl-2 gene product or polypeptide, a nucleic acid encoding a polypeptide encoding at least a portion of Bcl-2 gene product, nuclear matrix 23(nm23), a portion of nm23, a nucleic acid encoding a polypeptide comprising at least a portion of nm23, an apotosis-related protein, a portion of said protein, a nucleic acid encoding a polypeptide comprising at least a portion of the apoptosis-related protein, lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acid encoding a polypeptide comprising at least a portion of lipocalin NGAL, complement regulatory protein CD46, a portion of CD46, a nucleic acid encoding at least a portion of CD46, complement regulatory protein CD59, a portion of CD59, a nucleic acid encoding at least a portion of CD59, a nucleic acid encoding a portion of an FHIT gene, loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding at least a portion of MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding at least a portion of KAI1/CD82, a Fibroblast Growth Factor (FGF), a portion of FGF, a nucleic acid encoding a polypeptide comprising at least a portion of an FGF, Vascular Epithelial Growth Factor (VEGF), at least a portion of VEGF, a nucleic acid encoding at least a portion of VEGF, Insulin-like Growth Factor -1 (IGF-1), at least a portion of IGF-1, a nucleic acid encoding at least a portion of IGF-1, tumor amplified kinase STK15 (also BTAK and aurora2), at least a portion of STK15, a nucleic acid encoding a polypeptide comprising at least a portion of STK15, TMS-1, a portion of TMS-1, a nucleic acid encoding a polypeptide comprising at least a portion of TMS-1, maspin, at least a portion of maspin, a nucleic acid encoding a polypeptide comprising at least a portion of maspin, at least a portion of breast cancer associated (BRCA) gene, and at least a portion of a BRCA gene product; CDw60 protein, a portion of CDw60 protein or polypeptide, a nucleic acid encoding at least a portion of a CDw60 protein or polypeptide, mammary expressed enzymes including cytochrome P450s, catechol-Omethyltransferase, epoxide hydrolase, peroxidases, glutathione S-transferases, N-acetyltransferases, or sulfotransferases, a nucleic acid encoding at least a portion of a mammary expressed enzyme, Kallikrein 6 (zyme/protease M/neurosin) protein or polypeptide (hK6), a nucleic acid encoding at least a portion of an hK6 protein or polypeptide; and mammastatin protein or polypeptide, a nucleic acid encoding at least a portion of a mammastatin protein or polypeptide.
 5. A method as in claim 1, further comprising: examining the isolated ductal fluid to determine absorption of a molecule by abnormal cells in the fluid.
 6. A method as in claim 5, wherein the molecule comprises iodide.
 7. A method as in claim 1, further comprising: examining the isolated ductal fluid for a loss of heterozygozity.
 8. A method as in claim 1, further comprising examining the isolated ductal fluid for the presence of two or more markers.
 9. A method as in claim 1, further comprising examining the isolated ductal fluid for the absence of two or more markers.
 10. A method as in claim 1, further comprising examining the ductal fluid for the presence of at least one marker and the absence of at least one marker.
 11. A method as in claim 1, further comprising analyzing collected ductal epithelial cells by cytology.
 12. A method as in claim 9, wherein the markers are selected from the group consisting of DNA content, p53 gene or gene product, and G-actin or a nucleic acid encoding a polypeptide comprising at least a portion of G-actin.
 13. An isolated ductal fluid sample collected from a breast duct in a breast, said isolated ductal fluid not mixed with ductal fluid from any other breast duct.
 14. An isolated ductal fluid sample as in claim 13, wherein the sample comprises a marker for analysis.
 15. An isolated ductal fluid sample as in claim 14, wherein the analysis comprises determining the presence or absence of the marker.
 16. An isolated ductal fluid sample as in claim 13, a portion of said isolated ductal fluid not spontaneously discharging from the breast duct.
 17. An isolated ductal fluid sample as in claim 13, wherein the sample comprises 10 or more ductal epithelial cells.
 18. An isolated ductal fluid sample as in claim 13, wherein the sample comprises at least one ductal epithelial cells clump.
 19. An isolated ductal fluid sample as in claim 18, wherein the clump comprises 5 or more ductal epithelial cells.
 20. A method for analyzing breast markers or epithelial cells, comprising: determining the presence or absence of a marker in an isolated ductal fluid sample collected from a breast duct in a breast, said isolated ductal fluid not mixed with ductal fluid from any other breast duct.
 21. A method as in claim 20, wherein the duct from which the ductal fluid is isolated is not spontaneously discharging ductal fluid.
 22. A method as in claim 20, wherein the marker is selected from the group consisting of: lysophosphatidic acid, a lysophospholipid, paladin, a portion of palladin, a nucleic acid encoding a polypeptide comprising at least a portion of paladin, Lg, a portion of Lg, a nucleic acid encoding a polypeptide comprising at least a portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding a polypeptide comprising at least a portion of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, a nucleic acid encoding a polypeptide comprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid encoding a polypeptide comprising at least a portion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), a portion of REA, a nucleic acid encoding a polypeptide comprising at least a portion of REA, prothymosin alpha (PTA), a portion of PTA, a nucleic acid encoding a polypeptide comprising at least a portion of PTA, c-raf kinase, a portion of c-raf kinase, a nucleic acid encoding a polypeptide comprising at least a portion of c-raf kinase, CD66a, a portion of CD66a, a nucleic acid encoding a polypeptide comprising at least a portion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding a polypeptide comprising at least a portion of KL-1, cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acid encoding a polypeptide comprising at least a portion of CAM 5.2, leptin, a portion of leptin, a nucleic acid encoding a polypeptide comprising at least a portion of leptin, Bcl-2 gene product, at least a portion of Bcl-2 gene product or polypeptide, a nucleic acid encoding a polypeptide encoding at least a portion of Bcl-2 gene product, nuclear matrix 23(nm23), a portion of nm23, a nucleic acid encoding a polypeptide comprising at least a portion of nm23, an apotosis-related protein, a portion of said protein, a nucleic acid encoding a polypeptide comprising at least a portion of the apoptosis-related protein, lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acid encoding a polypeptide comprising at least a portion of lipocalin NGAL, complement regulatory protein CD46, a portion of CD46, a nucleic acid encoding at least a portion of CD46, complement regulatory protein CD59, a portion of CD59, a nucleic acid encoding at least a portion of CD59, a nucleic acid encoding a portion of an FHIT gene, loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding at least a portion of MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding at least a portion of KAI1/CD82, a Fibroblast Growth Factor (FGF), a portion of FGF, a nucleic acid encoding a polypeptide comprising at least a portion of an FGF, Vascular Epithelial Growth Factor (VEGF), at least a portion of VEGF, a nucleic acid encoding at least a portion of VEGF, Insulin-like Growth Factor -1 (IGF-1), at least a portion of IGF-1, a nucleic acid encoding at least a portion of IGF-1, tumor amplified kinase STK15 (also BTAK and aurora2), at least a portion of STK15, a nucleic acid encoding a polypeptide comprising at least a portion of STK15, TMS-1, a portion of TMS-1, a nucleic acid encoding a polypeptide comprising at least a portion of TMS-1, maspin, at least a portion of maspin, a nucleic acid encoding a polypeptide comprising at least a portion of maspin, at least a portion of breast cancer associated (BRCA) gene, and at least a portion of a BRCA gene product; CDw60 protein, a portion of CDw60 protein or polypeptide, a nucleic acid encoding at least a portion of a CDw60 protein or polypeptide, mammary expressed enzymes including cytochrome P450s, catechol-O-methyltransferase, epoxide hydrolase, peroxidases, glutathione S-transferases, N-acetyltransferases, or sulfotransferases, a nucleic acid encoding at least a portion of a mammary expressed enzyme, Kallikrein 6 (zyme/protease M/neurosin) protein or polypeptide (hK6), a nucleic acid encoding at least a portion of an hK6 protein or polypeptide; and mammastatin protein or polypeptide, a nucleic acid encoding at least a portion of a mammastatin protein or polypeptide.
 23. A method as in claim 20, wherein the marker is absorption of a molecule by abnormal cells in the fluid.
 24. A method as in claim 23, wherein the molecule comprises iodide.
 25. A method as in claim 20, wherein the marker is a loss of heterozygozity.
 26. A method as in claim 20, wherein the presence of two or more markers is determined.
 27. A method as in claim 20, wherein the absence of two or more markers is determined.
 28. A method as in claim 20 wherein the presence of at least one marker and the absence of at least one marker is determined.
 29. A method as in claim 20 wherein the marker determined is cytology of ductal epithelial cells.
 30. A method as in claim 20, wherein the marker is selected from the group consisting of DNA content, p53 gene or gene product, and G-actin or a nucleic acid encoding a polypeptide comprising at least a portion of G-actin. 